The long-term objective of this proposal is to understand the soluble methane monooxygenase (sMMO) system of proteins that converts CH4, O2 H+, and NADH selectively to CH3OH, H2O, and NAD+ under ambient conditions. Methanotrophs from which sMMO is isolated both prevent CH4, a greenhouse gas, from reaching the atmosphere and facilitate bioremediation. Oxidation of CH4 and other substrates is achieved in the hydroxylase enzyme (MMOH) at carboxylate-bridged non-heme diiron centers that also occur in related dioxygen-activating proteins. The sMMO proteins work in concert through the formation of complexes between MMOH, a reductase (MMOR), and an auxiliary protein (MMOB) that couples 02 consumption with CH4 oxidation. In order to understand the protein interactions, the complexes will be structurally characterized by Xray crystallography and advanced EPR methods that employ protein mutants bearing spin labels. The structure of MMOR will be determined by NMR spectroscopy. This information will be used to interpret the results of solution kinetics studies of electron-transfer (ET) from the terminal reductant, NADH, through MMOR to the diiron centers in MMOH. Individual steps of MMOR reduction of MMOH will be investigated by stopped-flow spectroscopy and by laser flash photolysis combined with spectroscopic monitoring of ET intermediates. The catalytic cycle of MMOH will be studied by single- and double-mixing stopped-flow and rapid freeze-quench methods in solution and by a novel cryoreduction approach in which the reduced enzyme generated in a frozen matrix is allowed to form oxygenated intermediates by annealing. EPR, ENDOR, Mossbauer, and EXAFS spectra will help characterize intermediates. Reactions of Q, the hydroxylating intermediate, with various substrates, in conjunction with analysis by advanced quantum mechanical techniques, will provide insight into the nature of the C-H bond activation step. MMOH will be expressed and mutants generated to test how specific protein residues assist the catalytic mechanism. The function of MMOD, a fourth protein component, will be studied by deleting it in the native organism and investigating its ability to help assemble the metal clusters in MMOH. Synthetic models of the diiron center in MMOH will be prepared. Their ability to hydroxylate tethered hydrocarbon substrates will be mechanistically studied. A new carboxylate-bridged diiron catalyst for oxo-transfer chemistry will be pursued. Dinucleating ligands that afford closer MMOH mimics will be employed to afford better hydrocarbon oxidation catalysts.